Eradication of solid tumors requires strategies that address the viable populations of malignant cells within hypoxic regions of such tumors. An insufficient and poorly organized vasculature, a major characteristic of rapidly growing tumor masses, results in poor oxygenation, high interstitial pressure, and a population of cells that are hypoxic, quiescent or slowly cycling, and distal to the blood supply, thus inadequate vascularization in solid tumors results in low oxygen and being difficult to reach with cytotoxic levels of drugs (Hockel, et al. Cancer Res. 1991, 51: 6098). Radiotherapy is thus ineffective in these areas as the radiation fails to generate sufficient oxygen radicals to result in cytotoxicity (Brizel, et al. Radiother Oncol. 1999, 53: 113). Moreover, the activity of cytotoxic drugs is also attenuated. Thus cells from these regions are frequently responsible for the re-establishment of disease. After treatment with oxygen-dependent cytotoxins such as x-irradiation, which generates oxygen radicals that damage cellular DNA, and conventional chemotherapeutics that target the well-oxygenated, rapidly growing portion of the tumor mass, the resistant hypoxic cell fraction can repopulate the tumor (Stratford, et al. Anticancer Drug Des. 1998, 13: 519). Moreover, hypoxic cells are subjected to an environment that enhances the selection of mutations which cause the progression of the neoplasm towards an increasingly aggressive phenotype. For example, hypoxia selects for cells deficient in p53-mediated apoptosis, enhances mutation rates, upregulates genes involved in drug resistance, angiogenesis, and tumor invasiveness (including HIF-1α), and thus is associated with a more metastatic phenotype (Ashur-Fabian, et al. Pro Natl Acd Sci USA. 2004, 101: 12236).
Prodrugs that act as hypoxia-selective cytotoxins generally must be substrates for one electron reductases such as NADPH:cytochrome (P450) reductase. The one-electron reduced prodrug radical, in the presence of oxygen, redox cycles back to the parent prodrug, preventing progression of the activation cascade and release of the cytotoxic, DNA damaging species. Under hypoxic conditions, further reduction of the radical anions alters the chemistry of the prodrug to allow release of the cytotoxic species (Yang, et al. Cancer Res. 2003, 63: 1520). Nitroaromatic and nitroheterocyclic compounds readily undergo one electron reduction to nitro radical anions (Korbelik, et al. Mutal Res, 1980, 78: 201). These molecules react rapidly with oxygen to regenerate the parental molecule. However in the absence of oxygen they are reduced further to generate hydroxylamine derivatives and then final aniline forms. While the nitro group is highly electron withdrawing, the hydroxylamine group is strongly electron donating. This results in a major change in the chemistry of the aromatic or heterocyclic ring, triggering the activation cascade and the release of parent drug.
As alkylating agents, a novel series of 1,2-bis(sulfonyl)hydrazine prodrugs (SHPs) with the ability to generate active chloroethylating species had been developed recently (Sartorelli, et al. U.S. Pat. No. 006,040,338, 2000; U.S. Pat. No. 005,637,619, 1997; U.S. Pat. No. 005,256,820, 1993; U.S. Pat. No. 005,214,068, 1993; U.S. Pat. No. 005,101,072, 1992; U.S. Pat. No. 004,849,563, 1989; and U.S. Pat. No. 004,684,747, 1987). The anti-tumor activity has been suggested to result from chloroethylating and subsequent cross-linking of DNA (Shealy, et al., J Med Chem. 1984, 27: 664).
1,2-Bis(methylsulfonyl)-2-(2-chloroethyl)-hydrazine carboxylic acid 1-(4-nitrophenyl)ethyl ester (KS119), the current lead compound in the SHP series, requires enzymatic nitro-reduction to generate the alkylating species 90CE, as demonstrated in FIG. 1. Thus, KS119 takes advantage of the hypoxic, reductive environment of solid tumors, thus creating an exploitable difference between cells in normal, well oxygenated tissues and hypoxic neoplastic cells (Shyam, et al. J Med Chem. 1999, 42: 941; and Seow, et al. Proc Natl Acad Sci USA. 2005, 102: 9282).
However, KS119 is rather insoluble in aqueous solution, even it has not sufficient solubility (<5 mg/mL) in co-solvent system like polyethylene glycol (PEG) and ethanol in order to meet clinical requirements of this drug. Therefore, our aim was to synthesize analogs of KS119 that (a) were capable of improving its water-solubility and stability in aqueous solution at pH 3 to 8; (b) were capable of forming chloroethylating species; and (c) were capable of maintaining hypoxia-selective activation.
Turning to the present invention, we believe that water-soluble compounds according to the present invention satisfy the above conditions. An example of such an SHP (KS119W) would be the phosphate-containing analog of KS119 shown in FIG. 2 for the following reasons:                (a) In general, a phosphate-bearing analog, including its salt form should have good water-solubility and stability at neutral pH;        (b) The bioconversion of compounds according to the present invention proceeds via alkaline phosphatase (AP) cleavage of the oxygen-phosphorous bond to form the phenol intermediate, as shown in FIG. 2.        (c) The bioconversion of the 2-nitrophenol intermediate is selectively activated under conditions of hypoxia to generate a hydroxylamine derivative or aniline form.        (d) The above intermediate of the amino analogs subsequently undergo fragmentation resulting in the formation of chloroethylating species (90CE). Release of 90CE would only occur on reduction of the nitro group under conditions of hypoxia.        (e) Compounds of the present invention are considered as prodrugs of 90CE that has been identified as an alkylating agent against a broad anticancer spectrum of neoplastic disease states, including, for example, numerous solid tumors.        